Degeneration of tissues with age is caused by their decreased renewal capacity over time. This results, at least in part, from irreversible growth arrest, or senescence, of the self-renewing stem cells that are responsible for tissue maintenance. Full understanding of senescence requires a comprehensive knowledge of all the genes responsible. Until recently, it was not possible to conduct a genome wide screen to identify these genes. However, the advent of short interfering (si) and short hairpin (sh) RNA technology to efficiently abolish gene expression, and its application to human cells, has now made such a screen possible. Extending our studies on the role of two chromatin regulators, HIRA and ASF1a, in cell senescence, we will perform a series of screens to identify genes that contribute to cell senescence. Specific Aim 1: To screen for shRNAs that abrogate senescence. Cell senescence is rapidly induced in primary cells by expression of oncogenes, such as activated Ras. We showed recently that chromatin regulators, HIRA and ASF1a, are likely effectors of Ras-induced senescence, and also induce senescence when ectopically expressed in primary cells. We will screen a virus-based, genome-wide library of shRNAs for those shRNAs that abrogate Ras- or ASF1a-induced senescence in human cells. Any shRNAs that abrogate senescence will implicate the corresponding cellular genes as inducers of senescence. Specific Aim 2: To verify that genes identified in Specific Aim 1 are novel regulators of senescence. We will use the following 3 criteria to verify that the genes identified in Specific Aim 1 are regulators of cellular senescence. We will ask: 1) whether they are required for cell senescence; 2) whether they are rate limiting for cell senescence; 3) whether the biology of the endogenous proteins is indicative of a role in senescence. In future studies, the genes identified in Specific Aim 1 and verified in Specific Aim 2, will be further investigated to define the molecular mechanism by which they contribute to senescence and, ultimately, to understand the molecular basis of age-dependent tissue degeneration. [unreadable] [unreadable] [unreadable]